Ischemic neonatal stroke is a common CNS disorder, yet no effective treatment is currently available. Embryonic stem (ES) cell transplantation after ischemic stroke has been tested extensively as a possible repair therapy in adult animals, however, little investigation has been done to evaluate ES cell transplantation in neonatal ischemic strokes. In the proposed studies, transplantation therapy using human ES cells (hESCs) will be tested in a rat neonatal ischemia model targeting the whisker-barrel cortex. We will address three key issues in hESC transplantation: 1) to promote survival of hESC- derived neural cells after transplantation into the harsh ischemic environment;2) to promote endogenous regenerative responses (angiogenesis and neurogenesis) after stroke and hESC transplantation, and to understand regulatory signals in these responses;3) to guide and improve synaptogenesis, neurovascular remodeling and functional recovery in the whisker-barrel pathway after hESC transplantation. Based on our previous investigations on mouse ES cells and preliminary studies on hESCs, neurally differentiated hESCs will be subjected to hypoxic preconditioning (HP) before transplantation in order to increase their tolerance to the destructive environment in the ischemic brain. Expression of the essential regulatory genes for cell survival and regenerative responses will be assessed after transplantation. Synaptic transmission represents a fundamental neuronal activity. We will test the hypothesis that HP can promote neurite outgrowth, synaptogenesis and functional activities of hESC-derived neurons in vitro (Aim 1) and in vivo (Aim 2). As a use-dependent rehabilitation strategy, intensified afferent signals induced by whisker stimulation will be tested for the ability to promote and guide transplanted hESC-derived cells and endogenous mechanisms in rebuilding better organized and more functional neurovascular architecture of the whisker-barrel cortex and thalamocortical connections (Aim 3). Cellular and molecular biological, immunohistochemical and electrophysiological techniques will be applied to acquire a comprehensive understanding of the mechanisms and functional benefits of human stem cell therapy boosted by HP and an enriched environment. Results from these experiments will likely improve the efficacy and efficiency of hESC transplantation in ischemic neonates.Transplantation therapy using human ES cells (hESCs) will be tested in a rat neonatal ischemia model targeting the whisker-barrel cortex. We will address three key issues in hESC transplantation: 1) to promote survival of hESC-derived neural cells after transplantation;2) to promote endogenous regenerative responses;3) to guide and improve the repair process and functional recovery in the whisker-barrel pathway after hESC transplantation.